The Anaerobic Endproduct Lactate Has a Behavioural and Metabolic Signalling Function in the Shore Crab Carcinus Maenas

The shore crab Carcinus maenas is often exposed to dramatic changes in its physico-chemical environment. These include daily, seasonal and spatial changes in, among other variables, oxygen concentration (2.8–67 kPa) and temperature (0–30 °C) (Morris and Taylor, 1983). Decapod crustaceans show many different physiological responses to environmental stressors (for reviews, see Mangum, 1983; McMahon and Wilkens, 1983; Vernberg, 1983). The physiological responses to hypoxia and temperature variation include changes in ventilation, circulation, haemocyanin oxygen-binding properties and in aerobic and anaerobic metabolism. However, information on circulating levels of biogenic amines in decapod crustaceans is scarce (Hoeger and Florey, 1989; Hoeger, 1990).

Lactate is the major anaerobic endproduct in crustaceans under hypoxic and anoxic conditions (Hill et al. 1991a,b). During a period of recovery from hypoxia, lactate levels are initially increased. Lactate is not excreted and only slowly remetabolised by gluconeogenesis (Hill et al. 1991a). Hill et al. (1991a,b) also showed that only part of the increased oxygen uptake associated with recovery from anoxia is concerned with the removal of lactate.

Besides physiological and metabolic adaptations, crustaceans also exhibit behavioural responses to hypoxia. The selection of a colder environment, i.e. behavioural hypothermia, has been described for crayfish Procambarus simulans in response to hypoxia (Dupré and Wood, 1988). Partial emersion under hypoxic conditions is also often observed in Carcinus maenas (Taylor et al. 1973, 1977; Taylor and Butler, 1973).

To elicit behavioural and physiological responses to adverse environmental situations, such as hypoxia, the animal must perceive this condition. The animal may sense this by a combination of several systems, i.e. peripheral or internal O₂ chemoreceptors, direct nervous response from the central nervous system or a response to circulating anaerobic endproducts. In the crayfish Astacus leptodactylus, the presence of peripheral O₂ receptors has been demonstrated (Massabuau and Burtin, 1984; Ishii et al. 1989). Wilkens et al. (1989) showed that parts of the nervous system of shore crabs, regulating ventilation and cardiac performance, are -sensitive, indicating the presence of a direct nervous response. The importance of a system in which anaerobic endproducts elicit responses from animals in adverse situations is much less clear. As far as we know, no information is available on the signalling function of lactate in crustaceans. Pörtner et al. (1994) recently found that lactate that is formed at O₂ levels below the critical and released into the extracellular fluid functions in the toad Bufo marinus as a metabolic alarm signal in conjunction with catecholamines, eliciting both behavioural and metabolic emergency responses similar to those observed under hypoxia. This signalling mechanism would imply a central action of lactate via the mediation of catecholamines. The presence of this function of lactate in endothermic animals such as mammals and birds or in invertebrates remains to be established (Pörtner et al. 1994).

The goal of this study was to test the hypothesis that a similar signalling function for lactate exists in marine crustaceans and to answer the following questions. Do shore crabs show behavioural hypothermia? Can lactate infusion elicit this behaviour? Does lactate infusion induce a change in the rate of oxygen consumption? Is this response modified by external ? How do the time courses of the responses to lactate infusion (behavioural and metabolic) correlate with lactate catabolism under different levels of external ? Is there a correlation between changes in lactate levels and changes in the levels of biogenic amines?

Shore crabs, Carcinus maenas (L.) (40–80 g; N=157), were obtained from commercial trawl-fishermen, who caught them in the German Bight of the North Sea in the autumn (September – November). After transport to the laboratory, they were held at densities of eight animals per 100 l in aquaria containing aerated recirculated North Sea water (35 ‰) at 13±0.5 °C and with a 12 h:12 h light:dark photoperiod. Crabs were kept in captivity for at least 2 weeks before experimental use. Nitrate, nitrite and ammonia levels in the sea water were checked at least weekly using sea water quality test kits (Tetra). Water was partially replaced when levels exceeded 6 mg l⁻¹ nitrate, 0.1 mg l⁻¹ nitrite and 0.25 mg l⁻¹ ammonia. Animals were fed mussels ad libitum twice a week. The crabs were starved for at least 24 h before experimental use. Only intermoult animals were used in the experiments.

To measure the preferred temperature, a temperature gradient was constructed in a rectangular aquarium, 2 m long by 0.25 m wide by 0.5 m high, covered with a lid. Three coils, made of stainless steel, were placed inside this aquarium, each connected to a powerful waterbath (2000 W, Lauda) for temperature control. To maintain a low minimum temperature in the gradient, the entire experimental apparatus was placed in a cold room at 0 °C. The gradient was linearised, vertical stratification was minimised and, to control ⁠, several aeration stones were placed on the bottom approximately 20 cm apart and the water was gently bubbled with either air or nitrogen delivered by a combination of mass flow controllers (MKS Multigas controller 647B GVS 19). The water depth was kept at 8 cm to maximise the stability of the gradient and to prevent the crabs from emerging from the water. The room was kept dark to exclude photophobic behaviour, and potential crab refuge sites were avoided in the aquarium design. The temperature gradient along the length of the aquarium was from 6 to 25 °C. For analysis of the preferred temperature, animals were equipped with temperature probes (Omega Pt100 micro-thermocouples). The probes were swiftly connected to the crabs by attaching them under a piece of rubber band previously glued to the dorsal carapace of the crabs using cyanoacrylate glue. The probe was connected to a MacLab data-acquisition system (MacLab 4E) to register water temperature continuously. Simultaneously, of the water was measured using a polarographic oxygen electrode (Eschweiler, Germany, no. 2000-100 4501). Data were sampled at 0.33 Hz and averaged over 1 min periods using the MacLab data-acquisition hardware and software (MacLab Chart V.3.3.3).

Two different experimental treatments were used. In the first treatment, animals were exposed to air-saturated sea water for 90 min, the gas mixture was then switched to nitrogen for 230 min and finally back to air for the remainder of the experiment (120 min). In the second treatment, animals that had been placed in the thermal gradient the night before were injected with a saline solution (460 mmol l⁻¹ NaCl, 13 mmol l⁻¹ KCl, 14 mmol l⁻¹ CaCl₂, 20 mmol l⁻¹ MgCl₂) after 90 min and a lactate solution 150 min later (460 mmol l⁻¹ sodium lactate, 13 mmol l⁻¹ KCl, 14 mmol l⁻¹ CaCl₂, 20 mmol l⁻¹ MgCl₂). The solution (500 μl) was injected into the base of the last walking leg. The composition of the solution was chosen to minimise the effects of fluctuating ion levels or an increase in proton level, which may influence catecholamine release, as has been reported for a hypoxic lactacidotic amphibian (Ambystoma tigrinum) (Talbot and Stiffler, 1991). To reduce the influence of potential circadian rhythms on the preferred temperature, all experiments were carried out at the same time of day.

The respiration measurements were performed using a flow-through respirometer (designed by F.-J.S. after Frier, 1976), placed in a thermostatted cold-room at 13 °C. This system (see Fig. 1) consisted of a maximum of six darkened respiration chambers connected to one polarographic oxygen electrode (Eschweiler, Germany, no. 2000-100 4501) via a rotating valve. During measurement of the outflow from a particular chamber, sea water was driven past the oxygen electrode by a peristaltic pump (Ismatec SW 5.01). Water flow through the five unconnected chambers was driven by gravity (at 80 ml min⁻¹). Incoming water, delivered from a constant-head tank, was bubbled with air or an air/N₂ mixture (⁠=12 kPa). Gas mixtures were delivered by a combination of mass flow controllers (MKS Multigas controller 647B GVS 19). Outflowing water was recirculated into the constant-head tank. Oxygen levels, either control values as found in the outflow of an empty chamber or experimental values from a chamber containing an animal, were measured with the oxygen electrode connected to an oxygen meter (Eschweiler M200). These data were sampled at 0.33 Hz using the MacLab data-acquisition hardware (MacLab 4E) and software (MacLab Chart V.3.3.3). Steady state was reached within 2 min after switching the electrode to a new chamber. Seawater flow over the electrode was not influenced by the flow range used, and flow was chosen such that the difference between inflowing and outflowing water was 10–15 % of that of the inflowing water.

To allow injection of saline or lactate solution, the animals (wet mass 64±12 g, mean ± S.E.M., N=13) were equipped with polyethylene catheters (i.d. 0.58 mm, o.d. 0.96 mm, length 30 cm) inserted in the coxa of the fifth pereiopod. Catheters were held in place with cyanoacrylate glue. To prevent loss of haemolymph, a latex membrane was glued to the coxa before the catheter was inserted through the epidermis. Surgery was conducted on submerged crabs and lasted for a maximum of 10 min. Animals were then placed into the darkened respiration chambers and allowed to recover and to adapt to the experimental chambers.

The experimental treatments used in the respiration experiments were as follows. In the first treatment, catheterised animals were acclimated in normoxic conditions to the respiration chamber for 45 h. At the end of the acclimation period, control rates of oxygen consumption in the animals were measured for at least 20 min. After this time, 500 μl of saline solution was injected, and the rate of oxygen consumption was measured for another hour. Approximately 24 h after saline injection, the rate of oxygen consumption was measured again before and after injection of the lactate solution.

In the second treatment, animals were tested under moderately hypoxic conditions. The was reduced to 12 kPa after 20 h, and animals were acclimated to hypoxia for another 25 h. Control rates of oxygen consumption in the animals were measured for at least 20 min. Then, 500 μl of saline solution was injected, and the rate of oxygen consumption was measured for another hour. Five hours later, the rate of oxygen consumption was again measured for 1 h. Approximately 24 h after saline injection, the rate of oxygen consumption was measured again before and after injection of the lactate solution. Lactate concentration in the haemolymph immediately after injection for both treatments was estimated to be 11.8±1.9 mmol l⁻¹ (mean ± S.D., N=13). This estimate, which assumes that the lactate remains in the haemolymph, is based on an estimated haemolymph volume of 30 % of the wet mass of the animal (Alspach, 1972; Gleeson and Zubkoff, 1977).

The metabolic removal of lactate from the haemolymph by catabolic processes (oxidation and gluconeogenesis) was studied by taking haemolymph samples over time from animals that had been injected with the lactate solution, under normoxic (>18 kPa) and moderately hypoxic (12 kPa) conditions. Individual crabs were isolated in darkened 2 l aquaria at 12±1 °C. Each aquarium was bubbled with either air or an air/N₂ mixture. After 15 h of acclimation to the aquarium, the first haemolymph sample was taken. One hour later, animals were injected with 500 μl of lactate solution at the base of the coxa of the first pereiopod. Haemolymph samples of approximately 0.5 ml were collected at the base of the last pereiopod at different times (0.25–36 h) after injection. Samples were collected in 1 ml plastic syringes using a hypodermic needle (0.6 mm×26 mm) inserted into the articular membrane at the base of the last walking leg. Samples that had not been collected within 20 s, including catching and handling, were discarded. Samples were immediately diluted in preweighed Eppendorf tubes containing 500 μl of ice-cold 0.28 mol l⁻¹ perchloric acid, and were weighed. Samples were then centrifuged for 4 min at 16 000 g and 2 °C (Eppendorf centrifuge 5402). The supernatant was frozen at −85 °C until analysis. Lactate concentrations were measured enzymatically (Bergmeyer, 1984).

Crabs were individually placed in darkened 2 l aquaria at 12 °C. The aquaria were placed in a large vessel under temperature control (Julabo FP30). Each aquarium was bubbled with either air or an air/N₂ mixture. After 15 h of acclimation to the aquarium under either normoxia (>18 kPa) or hypoxia (8.6±0.4 kPa), haemolymph samples (0.5–1 ml) were collected and treated as described above. The supernatant was divided between two Eppendorf tubes, frozen in liquid N₂ and stored at −85 °C until analysis for lactate (see above) or biogenic amines (see below). Three different experiments were performed. In the first, animals were subjected to a temperature decrease from 12 to 0 °C under normoxia. The second series investigated animals exposed to hypoxia at 12 °C, and the third series included animals exposed to hypoxia and to a temperature decrease from 12 to 0 °C. The temperature decrease from 12 to 0 °C took approximately 4 h. Samples were collected prior to and 3, 7, 15 and 24 h after the start of the temperature decrease (i.e. after 15, 18, 22, 30 and 39 h in the experimental aquarium).

Biogenic amine concentrations were determined in the supernatant of the same samples taken from animals exposed to low temperature and/or hypoxia (see above). The supernatant was thawed at 4 °C, diluted by a factor of between 4 and 6 with 0.1 mol l⁻¹ perchloric acid and filtered through a Millex-GV₄ filter (Millipore, 0.22μm). Samples were thawed and kept in the dark until analysis. HPLC with electrochemical detection was used for determination of biogenic amine levels. The isocratic chromatographic system consisted of a Pharmacia LKB 2249 pump with a constant flow of 1 ml min⁻¹, an injection valve (Rheodyne 9725i) with a 100 μl loop, a reversed-phase C18 column (CSC spherisorb ODS2, 5 μm, 25 cm×0.46 cm column) with a guard column (BioRad microgram ODS 5S) and an electrochemical detector (Chromsystems ECD 41000) with a glassy carbon electrode set to a potential of 1000 mV and a Ag–AgCl reference electrode. The mobile phase consisted of 75 mmol l⁻¹ NaH₂PO₄, 0.35 mmol l⁻¹ 1-octane-sulphonic acid, 10 μmol l⁻¹ EDTA, 9 % (v/v) methanol and 4 % (v/v) acetonitrile, pH 4.0, degassed and filtered through a 0.2μm nylon filter. Chemicals and amine standards were obtained from Sigma Chemicals. Calibration curves for standards were prepared from stock solutions kept in the dark at 4 °C. The amines analysed were noradrenaline, adrenaline, octopamine, dopamine, serotonin, tryptophan and tyramine. The compounds were identified by retention times of standard mixtures and by standard addition experiments. Amine concentration was calculated from the peak area compared with calibration curves for standard solutions (single standards and mixtures of standards) and checked by standard addition experiments.

Data were analysed using the software packages Statistica and SuperAnova and parametric (repeated-measures analysis of variance, ANOVA; contrast analysis; Pearson product correlation; Bartlett’s test for homogeneity of variances) and non-parametric (Spearman rank correlation; Mann–Whitney U-test; Kolmogorov two-sample test) techniques. Differences were considered significant at P<0.05.

Initially, shore crabs showed behavioural hyperthermia (Fig. 2). Animals placed in the temperature gradient consistently selected an initial preferred temperature of 19–20 °C, although their acclimation temperature was 12–13 °C. When the was reduced, the animals moved to a significantly lower temperature (Pearson product correlation test; r²=0.59; P<0.001). Exposure to anoxia resulted in a decrease in the preferred temperature of approximately 3 °C after 2 h. Return to normoxic values resulted in a significant (Pearson product correlation test; r²=0.57; P<0.001) increase in the preferred temperature. A return to the pre-treatment preferred temperature, however, was not accomplished within 2 h.

Animals injected with saline under normoxic conditions showed no significant change in their preferred temperature (repeated-measures ANOVA; P>0.05) (Fig. 3). When subsequently injected with lactate, they showed a significant (repeated-measures ANOVA with contrast analysis; P<0.01) decrease in preferred temperature. Although the response was transient, since the preferred temperature started to increase slowly after 30 min, pre-treatment preferred temperature was not reached within 4.5 h.

The rate of oxygen consumption doubled after C. maenas had been injected with saline or lactate (repeated-measures ANOVA with contrast analysis; P<0.05) (Fig. 4). Comparison of the responses to lactate and saline showed that there was no significant difference in the control rate of oxygen consumption, i.e. before injection. This indicates that the rates of oxygen consumption had returned to control levels within 24 h after saline injection, since lactate was injected 24 h after the saline injection. During the first 20 min after each injection, there was no difference in the rate of oxygen consumption between lactate-and saline-injected crabs. However, the rate of oxygen consumption 20 min after lactate injection was significantly (Kolmogorov two-sample test; P<0.05) elevated for the next 40 min compared with that of saline-injected animals. This means that oxygen consumption rates decreased faster after saline injection than after lactate injection.

During moderate hypoxia (⁠

Respiration rates during the first 60 min following injection did not differ between lactate-injected and saline-injected specimens (Fig. 5), but after 5 h, the rates of oxygen consumption of lactate-injected specimens were significantly higher (Kolmogorov two-sample test; P<0.05).

After 5 h, the rate of oxygen consumption of animals injected with saline was not significantly different from the pre-injection value (repeated-measures ANOVA; P>0.05). Another important difference between the two treatments is the difference in variability. Animals injected with lactate showed a clearly reduced variability compared with saline-injected animals (Bartlett; P<0.05).

Animals which had been injected with the same amount of lactate as in previous experiments (preferred temperature and oxygen consumption), showed a slow catabolism of haemolymph lactate. Initial lactate concentrations were calculated on the basis of a haemolymph volume of 30 % of the wet mass. These values, which also imply a homogeneous distribution of lactate in the whole haemolymph volume, were not significantly different from the values measured after 0.25 or 0.5 h (Mann–Whitney U-test; P>0.05). After 1 h, lactate levels under normoxia were significantly higher than those under hypoxia, possible because of the higher initial concentration (Mann–Whitney U-test; P<0.05) (Fig. 6). However, after 4 h, lactate levels under normoxia were significantly lower than those under hypoxia (Mann–Whitney U-test; P<0.05). This indicates that the rate of lactate utilization is higher under normoxia than under hypoxia. After 10 h, lactate levels for both normoxia- and hypoxia-exposed animals were not significantly different from control values (Mann–Whitney U-test; P>0.05).

Fig. 7 shows lactate concentrations in the haemolymph of animals exposed to a temperature decrease under normoxia, a temperature decrease under hypoxia and during hypoxia without a temperature change. A temperature decrease under normoxic conditions resulted in a significant (Mann–Whitney U-test; P<0.05) increase (2.7-fold) in lactate concentration after 7 h (0.37±0.19 mmol l⁻¹) compared with control values (0.14±0.12 mmol l⁻¹). After 15 h, lactate concentrations had returned to control values. No significant change in lactate level was noted during hypoxic exposure at 12 °C. However, a temperature reduction in hypoxic (8.6 kPa) animals resulted in a significant (Mann–Whitney U-test; P<0.01) increase (up to 36-fold) compared with control values. Maximum lactate levels (2.83±1.55 mmol l⁻¹) were reached after 15 h, after which time they decreased again, but they were still elevated (0.87±0.62 mmol l⁻¹) after 24 h compared with control values (0.09±0.06 mmol l⁻¹).

Fig. 8 presents the levels of the different biogenic amines detected in the haemolymph of animals exposed to hypoxia (8.6 kPa) or normoxia and a simultaneous temperature reduction.

A small but significant positive correlation was found between the levels of adrenaline, octopamine and tryptophan and the level of lactate (Spearman rank-correlation; r=0.36, P<0.01 for adrenaline; r=0.31, P<0.05 for octopamine, and r=0.30, P<0.05 for tryptophan; Fig. 9).

In comparison with control values (0 h), significant reductions (Kolmogorov–Smirnov two-sample test; P<0.05) were found in the levels of noradrenaline (3 h, 7 h, 24 h), and a significant increase (P<0.05) in the level of adrenaline (24 h) under normoxia in combination with temperature decrease (Fig. 8). Hypoxic animals showed a significant reduction in the levels of noradrenaline (24 h) and octopamine (24 h), and a significant increase in the levels of dopamine (7 h, 24 h) and tryptophan (3 h, 7 h, 24 h), after a temperature decrease.

Comparison of normoxic animals with hypoxic animals showed significant differences (Kolmogorov–Smirnov two-sample test; P<0.05) in the levels of noradrenaline (a decrease at 0 h, an increase at 24 h), adrenaline (an increase at 3 h, a decrease at 24 h), octopamine (an increase at 0 h, a decrease at 7 h), dopamine (an increase at 7 h), tyramine (a decrease at 7 h) and tryptophan (an increase at 7 h). Serotonin could be detected in only five of the 56 samples, and dopamine levels were also often below detection limits. The high level of inter-individual variability found in the biogenic amine levels in haemolymph samples should also be noted, together with the fact that noradrenaline (24 h) was not detected under normoxia, whereas dopamine was not detected during the hypoxic exposure experiment at 0 h, 3 h and 15 h.

The initial temperature selection by the crabs brought into the temperature gradient under normoxia may be explained in the light of findings with different crayfish species. In Procambarus spiculifer, P. clarkii and Cambarus latimanus, the preferred temperature in the laboratory matched the temperature in the natural environment encountered during the months of maximum physical activity (Taylor, 1984). Procambarus sp. also exhibited a circadian cycle in their preferred temperature matching the temperature cycles in their habitat. Acclimation temperature and starvation had no significant effect on the preferred temperature in several crayfish species (Crawshaw, 1983; Espina et al. 1993). The initial temperature selected by C. maenas also corresponds better with water temperature in the summer months than with the acclimation temperature (Morris and Taylor, 1983).

Shore crabs showed behavioural hypothermia when exposed to hypoxia. While this response is well known in both homeothermic and ectothermic vertebrates, this is only the second time that it has been described for an invertebrate (Dupré and Wood, 1988). The decrease in preferred temperature in the shore crab was of similar magnitude to that in a hypoxic goldfish Carassius auratus (Crawshaw et al. 1989), but much smaller than the decrease seen in the crayfish Procambarus simulans (Dupré and Wood, 1988). Behavioural hypothermia has several advantages for an aquatic ectothermic animal: a reduction in metabolic rate, a decrease in critical oxygen concentration, which is characterised by the onset of anaerobic metabolism (Pörtner and Grieshaber, 1993; Branco et al. 1993) and an increase in oxygen-binding capacity of the oxygen carrier with decreasing temperature. All of these responses increase the hypoxia-tolerance of the animal. Injection of shore crabs with lactate, resulting in a haemolymph lactate concentration similar to that seen under severe hypoxia (Lallier et al. 1987; Taylor et al. 1977; Hill et al. 1991b), also caused a decrease in the preferred temperature. Initially, this decrease occurred to a similar extent (3 °C) to that during severe hypoxia and to the same extent as the decrease found in Bufo marinus after injection of iso-osmotic sodium lactate (Pörtner et al. 1994). As with B. marinus, this finding supports the hypothesis that lactate alone can induce behavioural hypothermia and may act as a metabolic alarm signal.

The time course of the behavioural response to lactate injection may be interpreted as being related to the time course of normoxic lactate breakdown if it is assumed that there is a threshold concentration of lactate in the haemolymph above which lactate becomes effective at a central level (see below). The effect of temperature should be taken into account. Although the shore crabs did show behavioural hypothermia after lactate injection, the selected temperature was still 3.5 °C higher than the temperature at which the lactate breakdown experiment was performed, causing the lactate effect to disappear earlier in the behavioural than in the lactate breakdown experiment.

Additionally, the almost immediate onset of an increase in preferred temperature in animals which recover from severe hypoxic exposure may indicate that other mechanisms are also involved in regulating preferred temperature. Catecholamines may be a good candidate for contributing to the behavioural hypothermia response (see below). Alternatively, the oxygen-dependent catabolism of amines may be involved or O₂ sensors could affect the behaviour by sensing increased oxygen levels.

During both normoxia and moderate hypoxia, lactate injection resulted in increased rates of oxygen consumption (Figs 4, 5). The difference between the responses to saline and lactate injections indicates that lactate alone caused the long-term increase in rates of oxygen consumption. The difference between normoxic and hypoxic animals in the magnitude of the response, immediately after injection, is probably related to the larger aerobic metabolic scope at higher O₂ levels. The in these experiments was above the critical oxygen tension (Taylor et al. 1973, 1977) and does not result in anaerobic metabolism and lactate production. The rapid decline in the rate of oxygen consumption in normoxic animals may be related to the circulating level of lactate, although at first sig. ht lactate removal is too slow and poorly correlated with (Fig. 6). Also, the long-term response of hypoxic animals to lactate is probably due to the delayed decrease in lactate levels. The differences in time course can be explained if we assume that a minimum threshold level must be reached for lactate to become effective as a metabolic signal; this level may be higher than the 1.5 mmol l⁻¹ estimated for Bufo marinus (Pörtner et al. 1994). In accordance with this conclusion, haemolymph lactate level fell below the threshold level faster in normoxic than in hypoxic specimens. However, the data set is not as clear as that for Bufo marinus, and we cannot exclude the possibility that the lactate response may be more complex in C. maenas, where the threshold is either set at a higher level than in B. marinus or obscured by concentration gradients building up within the circulatory system.

The increase in rates of oxygen consumption after lactate infusion also contrasts with the typical pattern of oxygen consumption during progressive hypoxia in C. maenas. In this species, a decrease in ambient typically leads to .a slow and then, below the critical ⁠, sharp decrease in ⁠. C. maenas does not show the increase in rate of oxygen consumption at the critical that is seen in B. marinus (Pörtner et al. 1991), although both ventilation and cardiac output increase (Taylor, 1976; Taylor et al. 1977; Booth and McMahon, 1985). The response to lactate is smaller in hypoxic (above the critical ⁠) than in normoxic animals (Figs 4, 5), suggesting that, in contrast to the situation in B. marinus, the aerobic scope is too small in C. maenas to allow for an increase in rates of oxygen consumption when lactate production sets in as the critical is reached.

It might be argued that the process of lactate removal is responsible for the increase in metabolic rate. In crustaceans, oxidative catabolism and gluconeogenesis are the major routes for lactate removal (Hill et al. 1991a,b). Gluconeogenesis would only imply an increase in energy turnover. Any use of lactate in oxidative catabolism would therefore follow the energy requirements of the organism and would not, by itself, explain an increase in metabolic rate (the use of other substrates would consequently be reduced) (see Pörtner et al. 1994). This is supported by the findings of Hill et al. (1991a,b) who demonstrated that, during recovery from anoxia, only part of the increased oxygen uptake is associated with lactate removal. The increased rate of oxygen consumption during recovery from anoxia is not only of the same magnitude as that in lactate-injected animals but is also transient. These results also appear to contrast with the much slower time course of lactate removal (Booth and McMahon, 1985; Lallier et al. 1987; Hill et al. 1991a,b) unless, again, a threshold level is postulated above which lactate exerts its stimulatory effect.

An acute temperature decrease resulted in a small and transient, but significant, increase in haemolymph lactate levels. In contrast, moderate hypoxia at a constant temperature did not result in any lactate production. The combination of the temperature decrease with hypoxia resulted in a drastic increase in lactate level. This indicates that there is a synergistic effect of temperature decrease and hypoxia, emphasising that the ventilatory capacity and blood oxygen transport in crustaceans, as in some other ectothermic animals, may limit their ability to tolerate low temperatures (Mauro and Mangum 1982a,b; Zielinski and Pörtner, 1996; Sommer and Pörtner, 1996). Although the temperature in our experiments was very low, it is not outside the natural temperature range of the shore crab. Morris and Taylor (1983) reported water temperatures as low as 0.5 °C in rockpools at the Scottish coast, while the northern distribution limit of Carcinus maenas is the north of Norway and Iceland.

The positive correlation between lactate levels and the levels of adrenaline suggests that catecholamines may play a role in the emergency response. However, from these experiments, it is not possible to link directly changes in catecholamine levels with lactate-induced increases in rates of oxygen consumption and behavioural hypothermia. Nevertheless, these data are consistent with the results found for B. marinus, where a drenergic antagonists blocked the lactate-induced increase in ⁠. In goldfish, the presence of ethanol and the release of noradrenaline have been assumed to be linked, since both induce behavioural hypothermia (Wollmuth et al. 1987).

The significant correlations between both tryptophan and octopamine levels and lactate levels also suggest the involvement of monoamines in the lactate-induced emergency response. Changes in the levels of tryptophan, the precursor of serotonin, and octopamine can change the behaviour of crustaceans. Although serotonin was detected in only a few samples, it is known that serotonin induces tonic flexion of the tail muscle and aggressive behaviour in the lobster (Harris-Warrick et al. 1989). Octopamine induces muscle extension and behavioural submission. Monoamines are also known to modulate the sensitivity in the escape tailflip reflex in crayfish (Glanzman and Krasne, 1986). Both serotonin and octopamine are known to affect ventilation and circulation and may play a role in the response to handling stress in the shore crab (Wilkens et al. 1985; Wilkens and McMahon, 1992).

The results presented above indicate that lactate does indeed have a signalling function in crustaceans. This anaerobic endproduct helps the animal to sense unfavourable conditions. As such, it is an emergency signal which results in behavioural and metabolic changes, probably mediated by changes in biogenic amine levels. The behavioural change is a decrease in the preferred temperature of the animal, optimising its energy balance and oxygen transport. The signalling function seems to be performed independently of the ambient oxygen tension, but the metabolic response may be limited by the aerobic scope. Future experiments should include measurements of the threshold level of lactate required to elicit the metabolic and behavioural emergency response. Experiments using amine blockers and measurements of changes in the levels of biogenic amines after lactate infusion should be performed to confirm the role of biogenic amines in this emergency response model. The use of lactate as an anaerobic endproduct may be an evolutionary step forward compared with the use of other glycolytic endproducts such as opines, which are found in other invertebrate groups (Pörtner et al. 1994). The major advantage of lactate over opines is that lactate is distributed in a pH-dependent manner over different body compartments and accumulates predominantly in the body fluid, whereas opines are recycled intracellularly (Pörtner, 1993).

It still remains to be demonstrated that the signalling function of lactate exists in endothermic animals or in other lactate-forming invertebrate groups.